Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein Production and Purification
2.2. SEC-MALLS
2.3. Large Zone SEC
2.4. Analytical Ultracentrifugation
2.5. Small Angle X-ray Scattering (SAXS)
2.5.1. Data Collection and Model Free Analysis
2.5.2. Fit of a Polymer Physics Model to the Data
2.6. NMR Spectroscopy
2.7. X-ray Crystallography
3. Results
3.1. The Coiled-Coil Drives Tetramer Formation
3.2. A Dimer–Tetramer Equilibrium Exists at Sub-Micromolar Protein Concentrations
3.3. The Linker Sequence Can Be Modeled as A Worm-like Chain and Exhibits Only Weak Local Conformational Preferences
3.4. The Structural Transition between the Disordered Linker and the Ordered Binding Domain Is Not Abrupt
3.5. Crystallographic Model for the Interfacial Region Is Consistent with the NMR Observations
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Protein | |||
---|---|---|---|
“CC-L-BD” MenV P209-388 | “L-BD” MenV P267-388 | “L” MenV P267-328 | |
Instrument | Australian Synchrotron SAXS/WAXS beamline, Pilatus 1M detector | Australian Synchrotron SAXS/WAXS beamline, Pilatus 1M detector | Australian Synchrotron SAXS/WAXS beamline, Pilatus 1M detector |
Wavelength (Å) | 1.0332 | 1.0332 | 1.0332 |
q range (Å−1) | 0.01–0.60 | 0.01–0.60 | 0.01–0.60 |
Exposure time (s) a | 1 | 2 | 1 |
Protein concentration range (g/L) | 0.1–4.8 | 0.35–11.1 | 0.45–3.62 |
Buffer composition | 12.5 mM MOPS/KOH pH 7.0, 250 mM NaCl | 12.5 mM MOPS/KOH pH 7.0, 150 mM NaCl | 12.5 mM Tris/HCl pH 8.5, 150 mM NaCl |
Temperature (°C) | 10 | 10 | 10 |
SASDB accession codes | SASDLG9 | SASDLH9 | SASDLJ9 |
Crystal Form I | Crystal Form II | |
---|---|---|
Crystallization Conditions | ||
Protein concentration (μM) | 3300 | 3300 |
Crystallization Method | Sitting Drop Vapor diffusion | Sitting Drop Vapor diffusion |
Reservoir Solution | 1.65 M Ammonium Sulphate 0.20 M Malic acid/KOH pH 5.5 | 20% (w/v) PEG 5000 monomethyl ether 0.2 M PIPES/KOH pH 6.7 0.1 M Proline |
Temperature (°C) | 18 | 18 |
X-ray diffraction data | ||
Cryoprotectant | 1.65 M Ammonium Sulphate 0.20 M Malic acid/KOH pH 5.5 1M Lithium Sulphate 5 mM Maltose | 20% (w/v) PEG 5000 monomethyl ether 0.2 M PIPES/KOH pH 6.7 0.1 M Proline 20% (v/v) Ethlyene glycol 5 mM Maltose |
Space group | P21 | P212121 |
Unit cell dimensions | a = 57.6, b = 70.1, c = 111.7 Å β = 96.5° | a = 67.6 b = 77.4, c = 79.1 Å |
X-ray source | Australian Synchrotron Beamline MX1 | Rigaku MicroMax-007 HF Rotating Copper Anode |
X-ray wavelength (Å) | 0.95370 | 1.54179 |
Sample Temperature (K) | 110 | 110 |
Data resolution limits (Å) a | 59.26–1.31 (1.33–1.31) | 31.24–1.55 (1.57–1.55) |
Number of unique observations a | 149703 (307) | 59991 (2017) |
Mean Redundancy a | 3.2 (1.5) | 6.8 (5.6) |
Completeness (%) a | 70.7 (2.9) | 97.4 (66.5) |
Rmeasure a | 0.061 (0.857) | 0.026 (0.204) |
Rmerge a | 0.051 (0.607) | 0.026 (0.166) |
CC1/2 a | (0.783) | (0.982) |
Crystallographic models | ||
Number of protein molecules in the asymmetric unit | 2 × 431 residues | 1 × 431 residues |
Rwork/Rfree b | 14.9/19.6 | 16.7/19.0 |
Total number of protein atoms | 6998 | 3569 |
Number of water molecules | 1321 | 505 |
Other ligands | Maltose, Sulphate | Maltose, Ethylene Glycol, PIPES, Proline |
Disorder Model | Individual Anisotropic B-factors | Individual Isotropic B-factors |
Mean total isotropic B-factor, all protein atoms (Å2): | 11.7 | 15.0 |
Bulk Solvent model | Mask | Mask |
RMSD from ideal geometry: Bond lengths (Å)/Bond angles (°) | 0.009/1.495 | 0.010/1.398 |
Residues in Favoured/Allowed regions of Ramachandran plot (%) c | 99/1 | 99/1 |
PDB ID | 7KD4 | 7KD5 |
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Webby, M.N.; Herr, N.; Bulloch, E.M.M.; Schmitz, M.; Keown, J.R.; Goldstone, D.C.; Kingston, R.L. Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions. Viruses 2021, 13, 1737. https://doi.org/10.3390/v13091737
Webby MN, Herr N, Bulloch EMM, Schmitz M, Keown JR, Goldstone DC, Kingston RL. Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions. Viruses. 2021; 13(9):1737. https://doi.org/10.3390/v13091737
Chicago/Turabian StyleWebby, Melissa N., Nicole Herr, Esther M. M. Bulloch, Michael Schmitz, Jeremy R. Keown, David C. Goldstone, and Richard L. Kingston. 2021. "Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions" Viruses 13, no. 9: 1737. https://doi.org/10.3390/v13091737